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Mutagenesis, Vol. 14, No. 1, 77-82, January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press

Detection of the `4977 bp' mitochondrial DNA deletion in human atherosclerotic lesions

Massimo Bogliolo1, Alberto Izzotti2,3, Silvio De Flora2,6, Carla Carli4, Angelo Abbondandolo1,5 and Paolo Degan1

1 Laboratory of Mutagenesis-CSTA, Istituto Nazionale per la Ricerca sul Cancro, Largo R.Benzi 10, I-16132 Genoa, 2 Institute of Hygiene and Preventive Medicine, University of Genoa, Via A.Pastore 1, I-16132 Genoa, 3 Centro Interuniversitario per la Ricerca sul Cancro, University of Genoa, Largo R.Benzi 10, I-16132 Genoa, 4 Anatomic Pathology Department, Sampierdarena Hospital, Corso Scassi 1, I-16149 Genoa and 5 Department of Clinical and Experimental Oncology, University of Genoa, Largo R.Benzi 10, I-16132 Genoa, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of the 4977 bp deletion (`common deletion') in the mitochondrial DNA (mtDNA) is associated with defects in the metabolic machinery acquired during ageing as a hallmark of a degenerative phenotype. We analysed 27 samples (18 from surgical patients and nine from autopsy cases) of DNA extracted from smooth muscle cells of abdominal aorta fragments affected by atherosclerotic lesions. The deletion was detected by PCR amplification–gel electrophoresis and characterized by sequencing of the PCR product. The mtDNA `common deletion' was detected in all analysed samples. However, its levels were not particularly high, which may be ascribed to the fact that smooth muscle cells in atherosclerotic lesions have a lower energy requirement and an appreciable proliferation rate, as compared for instance with cardiac myocytes. When the subjects were divided into two numerically equivalent age classes (60–72 years plus a 45-year-old subject versus 73–95 years), the deletion had significantly higher levels in the older subjects. Conversely, its presence did not correlate with source (surgical or autoptic), sex, cigarettes consumption, other clinical and anamnestic parameters or with the levels of adducts and 8-hydroxy-2'-deoxyguanosine measured in the nuclear DNA of the same samples. A previously unreported deletion of 5111 bp was additionally found in the mtDNA from a 45-year-old woman. The origin of this lesion seems to be compatible with the slipped mispairing model proposed for the `common deletion'.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
During the last two decades evidence has been provided that specific mutations and aberrations in the mitochondrial DNA (mtDNA) of somatic cells are associated with inheritable diseases (Wallace, 1992Go). mtDNA deletions and duplications are involved in clinically complex pathological manifestations such as Pearson's syndrome (Rotig et al., 1995Go), Kearns–Sayre syndrome (Zeviani et al., 1988Go), chronic progressive external ophtalmoplegia (Degoul et al., 1991Go) and several other syndromes in which the patient phenotype, although normal at birth, leads to a premature and distressful ageing process (Wallace, 1992Go; Lestienne and Bataille, 1994Go; De Flora et al., 1996Go). These discoveries stimulated the search for possible `spontaneous' mtDNA mutations and deletions in ageing. Many deletions of variable extension were found. These alterations accumulate with age in post-mitotic cells with a strikingly characteristic tissue specificity (Lestienne and Bataille, 1994Go). A specific deletion of 4977 bp, called the `common deletion' (mtDNA4977), is detected at higher frequency in adult brains and hearts whereas it is undetected in fetal and young tissues. This deletion accumulates much faster in tissues with high metabolic rate and low proliferative index (Cortopassi et al., 1992Go; Wallace, 1992Go).

The free radical theory of ageing suggests that the accumulation of potentially harmful side-products of oxygen metabolism may well contribute to the progressive deterioration of cells and tissues with time (Holmes et al., 1992Go). The proximity of mtDNA to the site of generation of reactive oxygen species contributes to a higher rate of damage to mtDNA, as compared with nuclear DNA (Imlay and Linn, 1988Go; Balansky et al., 1996Go; De Flora et al., 1996Go). As a consequence, mitochondrial genes coding for oxidative phosphorylation enzyme complexes, mainly those coding for Complex I, are damaged at high frequency, which results in a reduced capacity for energy production and enhanced production of superoxide anions (Cortopassi and Wang, 1995Go). These findings lead to the hypothesis that mitochondrial degeneration could be linked to the ageing process by contributing to the drift from physiological parameters to biochemical alterations of pathological significance (Arnheim and Cortopassi, 1995). This drift may affect, more or less extensively, either the precocity or the clinical relevance of the pathological process.

Atherosclerosis provides an ideal system to study the role of oxygen radicals in an ageing process of complex and multifactorial origin. Focal intimal thickening in the early phases of the development of atherosclerotic lesions involves accumulation of cells, lipids and connective tissue that results in episodic ischemia associated with the generation of reactive oxygen species (Ross, 1993Go; Witztum, 1994Go; Ramos et al., 1996Go). During this process, the electron transport chain is inhibited, thereby resulting in a diminished adenine nucleotide pool, increased electronegativity, oxidation of low density lipoproteins and depletion of scavengers, with consequent mitochondrial damage, tissue injury and necrosis. Cell death is a prominent component of the human atherosclerotic plaque. Furthermore, active cell depletion is stimulated through apoptosis by the presence of high concentrations of cytokines (Witztum, 1994Go; Bennett et al., 1995Go; Geng and Libby, 1995Go).

The presence of high levels of mtDNA4977 in cardiac muscle from patients suffering from coronary atherosclerotic heart disease has been documented (Corral-Debrinski et al., 1991Go, 1992Go). The aim of the present study was to test for the presence of this mtDNA damage in human abdominal aorta specimens affected by atherosclerotic lesions. In addition, frequency and levels of mtDNA deletion were related to exposure of the subjects to atherogenic risk factors and were compared with the levels of adducts and oxidative damage to nuclear DNA, as evaluated in the same samples tested in a parallel study (De Flora et al., 1997Go).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients and samples
Table IGo summarizes the characteristics of the individuals under study. Fragments of abdominal aorta containing atherosclerotic lesions were removed at surgery from 18 patients undergoing aortic graft due to severe aneurysm of the sub-renal aorta at the Galliera Hospital, Genoa, Italy (De Flora et al., 1997Go). Nine additional samples of abdominal aorta were collected at autopsy 24 h after death at the Sampierdarena Hospital, Genoa, Italy. Irrespective of its source, each aorta sample was almost entirely derived from a fragment containing atherosclerotic lesions. Immediately after removal, the aorta fragments were immersed in sterile physiological saline solution, stored at 4°C and transferred to the Institute of Hygiene and Preventive Medicine of the University of Genoa.


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  		Table I. Source and characteristics of abdominal aorta samples used in the present study and results relative to detection and quantification of the mtDNA 403 bp band (4977 `common deletion')
 
DNA extraction
Each fragment was examined and classified according to its macroscopical appearance and carefully cleaned of calcifications, fatty deposits and thrombotic material. After washing with physiological saline the three artery layers were dissected and separated as accurately as possible. The tunica media, which is mainly composed of smooth muscle cells (SMC), as confirmed by histological analyses (De Flora et al., 1997Go), was used for total (genomic and mitochondrial) DNA extraction. Within 1 month of storage at –80°C the tunica media was homogenized in a Polytron apparatus at 4°C in 250 mM sucrose, 50 mM Tris–HCl, pH 7.4. DNA was isolated by solvent extraction using an automatic DNA extractor (Genepure 341; Applied Biosystem, Foster City, CA). The procedure for DNA extraction was basically as described by Gupta (1984), except that the homogenized tissue was sequentially treated first with a mixture of RNases A and T1 for 1 h and then with proteinase K for 5 h at 55°C, followed by two extractions with a phenol/water/chloroform mixture and two extractions with chloroform only. The extracted DNA was quantified by spectrophotometric analysis. The whole procedure was performed under a helium atmosphere and using phenol of the highest available quality in order to avoid auto-oxidation phenomena.

Analysis of mitochondrial DNA and quantification of the 4977 bp `common deletion'
These analyses were carried out at the Laboratory of Mutagenesis-CSTA, Istituto Nazionale per la Ricerca sul Cancro, Genoa. In order to estimate the presence and the amount of mtDNA4977, aortic DNA was serially diluted and each dilution was tested by means of PCR for the presence of total and deleted mtDNA. The first set of primers (forward, 4504–4526 5'-ccatctttgcaggcacactcatc-3'; reverse, 4977–4955 5'-atccacctcaactgcctgctatg-3') (TIB MolBiol, Genoa, Italy) yields a 473 bp DNA fragment upon amplification of a region near the replication origin of the mitochondrial genome, which is known not to be susceptible to deletions (Hamblet and Castora, 1995Go). This fragment was used for quantification of total mtDNA. If mtDNA with the `4977' deletion is present, the forward 8282–8305, 5'-cccctctagagcccactgtaaagc-3', and reverse 13662–13643, 5'-gttagtaagggtggggaagc-3', primer set yielded a 403 bp fragment after amplification.

Prior to performing PCR, ~2 µg of aortic DNA were digested overnight at 37°C with 2 U PstI and 2 U EcoRI which, within the deleted region, cut at positions 9020 and 12640, respectively. This procedure avoids amplification of wild-type products during the PCR reaction with the forward 8282–8305 and reverse 13662–13643 primer couple. Serial dilutions of 2 µg of PstI + EcoRI-digested DNA were performed to yield DNA concentrations in the range 0.050–50 ng for amplification of the total mtDNA and 62.5–500 ng for the deleted mtDNA.

PCR was performed using 50 pmol of each primer, 250 mM each dNTP, 50 mM KCl, 10 mM Tris–HCl, pH 8.3, 1.5 mM MgCl2, 0.1% Triton X-100 and 1 U DyNAzimeTM thermostable DNA polymerase (Fynnzymes Oy, Riihitontuntie, Finland), in a final volume of 50 µl. A negative, DNA-free control was prepared for every set of PCRs, which were performed in a MJPT-100 thermal cycler (MJ Research, Watertown, MA) for 35 cycles. Each cycle consisted of 1 min at 94°C, 1 min at 63°C and 2 min at 72°C for both primer sets. The whole amplification mixture was electrophoresed on 2% agarose (BioRad, Richmond, CA) in TAE gel incorporating ethidium bromide to a final concentration of 0.5 mg/ml.

The gels were photographed under UV transillumination with 4x5 inch Polaroid 55 films and the negatives were analysed by means of a GS-300 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). With all samples, the decline in optical density (OD) of the PCR products for each dilution series versus the amount of mtDNA used in the PCR reactions fitted a semi-logarithmic plot, according to a regression line identified by the equation OD = a + bxlog(ng DNA) (Figure 1Go), with r2 > 0.90. The percentage of deleted mtDNA with respect to total mtDNA was determined by the ratio of the DNA dilutions that reduced the PCR product OD of the deleted mtDNA and total mtDNA curves to the same level (Corral-Debrinski et al., 1991Go). An arbitrary threshold of 0.0037% was assumed, since the correlation between OD and log(ng DNA) was not linear below this level under our experimental conditions. To confirm the nature of the PCR products obtained from samples showing the presence of deleted mtDNA, all PCR reaction products were sequenced on both strands with an ABI PRISMTM Dye terminator cycle sequencing Ready Reaction Kit (Perkin-Elmer, Milano, Italy) on an Applied Biosystems 377 Automated Sequencer (Perkin-Elmer, Milano, Italy), after purification with Microcon 100 (Amicon, Beverley, MA). Data were analysed with InStat software (GraphPad Software, San Diego CA).



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  		Fig. 1. (A) Example (sample A87) of quantification by serial dilution-PCR of total and deleted aliquots of aortic smooth muscle cell mtDNA; 473 and 403 bp bands, whose intensities are related to mtDNA amounts, are visible in the lanes corresponding to total mtDNA and deleted mtDNA, respectively. The 403 bp fragment indicates the presence of the 4977 bp `common deletion'. The band lower than 100 bp corresponds to residues of the DNA primers used for PCR. Negative controls, i.e. DNA-free samples undergoing PCR (see Materials and Methods) are identified as Total mtDNA NC and Deleted mtDNA NC. (B) Densitometric analyses of total mtDNA versus 4977 bp mtDNA deletion.

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The characteristic PCR product (403 bp) for the mtDNA4977 deletion was detected after gel electrophoresis in all analysed samples. As shown in Table IGo, six of the 18 surgical samples (33.3%) had deletion levels ranging from 0.0078 to 0.16% of the total mtDNA (mean ± SD 0.045 ± 0.06%). Five of the nine autopsy samples (55.6%) had deletion levels ranging from 0.0037 to 0.134% of the total mtDNA (mean ± SD 0.049 ± 0.05%). The difference between surgical and autopsy samples showing proportions of deleted mtDNA higher than the arbitrary threshold limit or their mean levels were not statistically significant, as evaluated by Fisher's exact test and Student's t-test, respectively. There was no sex-related difference, the mtDNA4977 being >0.0037% in eight of 20 males (40.0%) and three of seven females (42.9%).

By sequencing analysis we confirmed the identity of the mtDNA4977 deletion for all samples and found that the deletion itself involves two 13 bp direct repeats. One autopsy sample from a 45-year-old woman (A103C) showed a second product of 269 bp besides the `normal' 403 bp product after amplification with the 8282 forward and 13662 reverse primers (Figure 2AGo). This second PCR product, characterized by DNA sequencing, appeared to be generated by a novel deletion of 5111 bp from position 8467 to 13590 of the mitochondrial genome (Figure 2BGo).



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  		Fig. 2. (A) PCR products of aortic smooth muscle cell mtDNA from a 45-year-old women (sample A103C) after serial dilution amplification. Besides the 473 bp PCR product related to total mtDNA and the 403 bp band related to the 4977 bp `common deletion`, a 269 bp band is evident, which is related to a 5111 bp deletion of mtDNA. (B) Sequences flanking the two involved direct repeats, consisting of 13 bp for the mtDNA `common deletion' and 10 bp for the novel one.

 
As shown in Figure 3Go, the mtDNA4977 was >=0.0037% in three of 13 subjects aged <72 years (23.1%) and in eight of 13 subjects aged >72 years (61.5%). This qualitative difference was statistically significant (P < 0.05), as assessed by {chi}2 analysis. Also, from a quantitative standpoint the difference between the two age classes was statistically significant (P < 0.05), as assessed by the non-parametric Mann-Whitney test.



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  		Fig. 3. Relationships between age of subjects and mtDNA4977 deletion frequency and levels in smooth muscle cells from abdominal aorta affected by atherosclerotic lesions. Open circles refer to surgical samples and closed circles refer to autopsy samples. The vertical dashed line corresponds to the median age of the examined subjects (72 years). The horizontal dashed line corresponds to 0.0037% mtDNA4977 deletion, which was assumed as a threshold of sensitivity under our experimental conditions.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The aim of the present study was to acquire data on the presence of possible alterations in the mtDNA of aorta SMC samples obtained from atherosclerotic lesions. The presence of the mtDNA4977 deletion was detected by PCR analysis in all examined samples, although appreciably high levels of it were only found in 33.3% of the surgical samples and in 55.6% of the autopsy samples. As previously reported (Schon et al., 1989Go), this deletion appears to be generated through a process of slipped mispairing from two 13 bp direct repeats.

A new mtDNA deletion was characterized in the mtDNA from one autopsy sample, obtained from the only relatively young subject under study, who died due to heart failure. This deletion is localized between nucleotides 8467 and 13590 and the regions that involve this deletion and the 4977 bp deletion almost overlap. This novel deletion is also likely to be generated by a slipped mispairing process, similar to that proposed for mtDNA4977, between two 10 bp direct repeats (5'-ctacctccct-3') at positions 8468–8477 and 13580–13589.

The frequency of the mtDNA4977 deletion in clinically normal hearts accumulates with age in individuals older than 30 years (Corral-Debrinski et al., 1992Go). Our data show that even within elderly subjects, there is an increase with age of this deletion in smooth muscle cells from atherosclerotic lesions. In fact, by dividing the subjects into two numerically equivalent age classes, in subjects aged >72 years the mtDNA4977 deletion was three times more frequent and had significantly higher levels as compared with subjects aged <72 years, even when including in the latter group the sample of the 45-year-old woman positive for both the mtDNA4977 and mtDNA5111 deletions.

Relatively low amounts of mtDNA4977 were detectable in these samples of aorta smooth muscle cells, as compared with the amounts found in cardiac muscle mtDNA from a population of individuals suffering from coronary atherosclerotic heart disease (Corral-Debrinski et al., 1991Go, 1992Go). These differences can presumably be ascribed to the different tissues examined in the two studies. In fact, mtDNA4977 is known to accumulate much faster in tissues with a high metabolic rate and low proliferative index (Wallace, 1992Go). Cardiac myocytes are perennial and fully differentiated cells in adults and have a high energy requirement. It has even been found that within patients suffering from coronary atherosclerotic heart disease, the left ventricle, having a higher work load and energy requirement, accumulates 50-fold more mtDNA damage than the right ventricle (Corral-Debrinski et al., 1992Go). Arterial smooth muscle cells have a lower energy requirement and their proliferation rate is low under normal conditions (Gordon et al., 1990Go), but can be stimulated during the atherogenic process by certain factors, such as platelet-derived growth factor (Ross, 1986Go) and lipoprotein(a) (Grainger et al., 1993Go). Moreover, smooth muscle cell mitogens and chemotactic agents are released during regenerative repair processes in injured cells and mutations could induce constitutive production of growth factors in these cells, thereby resulting in an autocrine stimulation of growth (Ramos et al., 1996Go).

The population of 18 surgical samples that was examined in this study is a subset of a larger population of 84 samples that had been previously assayed for an exhaustive evaluation of different molecular biomarkers in nuclear DNA (De Flora et al., 1997Go). The samples tested for the mtDNA4977 deletion were selected based on the amounts of DNA remaining from the previous analyses and on the availability of data on 8-hydroxy-2'-deoxyguanosine (8-OH-dG). 32P-post-labelling analyses were positive in all tested samples, showing up to nine individual DNA adducts, with total levels ranging from 5.9 to 51.0 adducts/108 nucleotides. Statistically significant correlations were found between the levels of 32P-post-labelled DNA adducts and atherogenic risk factors, including age, the number of currently smoked cigarettes per day, the ratio of total to high density lipoprotein blood cholesterol, blood triglycerides and blood pressure, as well as synchronous fluorescence spectrophotometry-positive adducts and oxidative DNA damage, measured as 8-OH-dG (De Flora et al., 1997Go). In contrast, no correlation was found between DNA adduct levels or atherogenic risk factors and the amounts of the mtDNA4977 deletion. Also, there was no significant correlation between the levels of 8-OH-dG measured in 10 of the 18 samples (55%) and amounts of the mtDNA4977 deletion.

In conclusion, the mtDNA4977 deletion is consistently detectable in the mtDNA of smooth muscle cells from atherosclerotic lesions in elderly subjects. However, its levels are not particularly high and, at least in the examined samples, do not appear to be related to the intensity of oxidative damage affecting the nuclear DNA of the same cells.


   Acknowledgments
 
We thank Prof. G.L.Petrilli for supplying the surgical aorta samples and Dr A.Torroni and Mrs Silvia Borghini for their technical skills. This work was partially supported by the Italian Ministry for University and Scientific-Technologic Research (MURST), the Genoese Athenaeum Project and the CNR Targeted Project ACRO.


   Notes
 
6 To whom correspondence should be addressed. Tel: +39 10 353 8500; Fax: +39 10 353 8504; Email: sdf{at}unige.it Back


   References
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 Introduction
 Materials and methods
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 Discussion
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Received on March 25, 1998; accepted on August 7, 1998.


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